In middle and late adulthood, all people experience a series of progressive alterations in body physiology and composition. One physiological change associated with aging is immune system senescence. This senescence is characterized by a decline in both B-cell (humoral) and T-cell (cellular) immune responses. B-cells and T-cells are collectively known as lymphocytes. In basic terms, the B-cell immune response is responsible for the production of specific antibodies targeted to specific antigens such as bacteria and viruses. The T-cell immune response is responsible for the production of activated T-cells which recognize and destroy foreign tissues and cells infected with pathogens. It also produces lymphokine secreting helper T-cells that stimulate the bodies overall immune response.
The decline in the immune response with age is thought to be a major contributor to the relatively higher susceptibility of elderly people to infectious diseases and to cancer. Of the infectious diseases, influenza infection is a major health problem causing death in a significant percentage of elderly people contracting the disease each year. The institution of vaccination programs to protect the elderly from influenza have been only partially successful, mainly because the elderly are significantly less likely to develop a protective antibody response to the influenza vaccines than younger people. Thus, the development of a safe, effective and convenient method for enhancing the efficacy of vaccines would be an important step forward in the field, particularly influenza vaccines, of preventive medicine.
Another physiologic change associated with aging is the decreased oxidation of fatty acids. This metabolic process, which is a major source of cellular energy, takes place in the mitochondria. Before most fatty acids can be metabolized, however, they must be esterified with L-carnitine to form acylcarnitine. The fatty acid acylcarnitine can then be transported across the mitochondrial membrane. Once inside the mitochondria, the acyl group is transferred to Coenzyme A. The carnitine is then transported back out of the mitochondria either in its free form or is esterified to acyl groups needed to be removed from the organelle. Thus, carnitine has two critical functions in the cell, namely, (1) to stimulate fatty acid oxidation by transporting acyl groups across the inner mitochondrial membrane, resulting in adenosine triphosphate (ATP) formation, and (2) to remove extra or "toxic" acyl groups from the mitochondria and cell as carnitine esters.
L-carnitine is present in serum and urine in both free and esterified forms. In humans, normal total serum carnitine concentrations range from about 25-79 .mu.M, and free carnitine concentrations range from about 21-68 .mu.M. One indication of the level of fatty acid oxidation in an individual is the ratio of acylcarnitine (esterified carnitine) to free L-carnitine in these fluids, referred to as the E/F ratio. An E/F ratio greater than 0.25 is indicative of reduced oxidative activity; therefore, it is not surprising that 65% of adults over the age of 65 have an E/F ratio exceeding 0.25 since, as mentioned above, fatty acid oxidation tends to be reduced in this population of people.
In addition to its role in the oxidation of fatty acids, L-carnitine has also been found to exhibit some immunomodulatory effects in vitro. For example, Franceschi C. et al. showed that phytohaemagglutinin-induced peripheral blood lymphocyte proliferation was markedly increased by acetyl-L-carnitine-preloaded lymphocytes from young and especially old subjects. Int. J. Clin. Pharm., Res. X (1/2) 53-57 (1990). In addition, U.S. Pat. No. 4,415,588, issued to Cavazza, discloses that the incubation of mouse and human lymphocytes with L-acetylcarnitine and phytohemagglutinin or concanavalin A in vitro, resulted in a larger proliferative response than that seen if the mitogens were used alone.
These reports, while important observations, leave many questions unanswered. For example, they are all in vitro studies and, therefore, it is unknown whether their effects in vitro would also occur in vivo. Also, there was no characterization of which population of lymphocytes, the B-cells or T-cells, were proliferating in response to the effectors.
Vaccines function by introducing antigens into the body which can then bind to the surface antibodies of circulating B-cells. The binding of the antigen to the B-cell surface antibody activates the B-cell causing it to proliferate and synthesize and secrete antigen-specific antibodies. Mitogens can also cause B-cells to proliferate, however, unlike the proliferation generated by an antigen-antibody interaction, the proliferation of B-cells caused by mitogens is independent of their ability to produce antibodies. Unless a B-cell is already activated to produce a specific antibody, a proliferative response generated by a mitogen will not result in increased production of specific antibodies by the cell. Further, even if a B-cell is already activated, the mitogen induced proliferation may or may not stimulate greater antibody production by the cells.
For an immunomodulator to be of use as an adjuvant, it would have to increase the antigenicity of the vaccine, increase the production of antigen-specific antibodies by activated B-cells, and/or increase the rate of proliferation of the activated B-cells.
Some of the most potent adjuvants presently known cannot be used in clinical practice because they are either toxic or cause potentially fatal hypersensitivity reactions in animals after several exposures. These include Freund's adjuvant, incomplete Freund's adjuvant, and keyhole limpet hemocyanin.
Other adjuvants with either potential or proven clinical application fall into two categories; those that are delivered with the vaccine antigen as part of the injection, and those that are administered separately.
Those adjuvants falling within the first category include alum, gram negative bacterial derivatives, mycobacterial derivatives, and lipid derivatives. Alum has no serious side effects but is considered a very weak adjuvant. The gram negative bacterial derivatives, including monophosphorylated derivative A (MPL), Bacille Calmette-Guerin (BCG), and lipopolysaccharide (LPS) are potent adjuvants. However, they may uncouple T-cell regulation of B-cells and there is currently concern that they may, therefore, have carcinogenic potential. The mycobacterial derivatives such as trehalose dimycolate (TDM) and cell wall cytoskeleton (CWS) have been used successfully as adjuvants only in conjunction with MPL. Therefore, the above concerns regarding the use of MPL also apply to these compounds. The lipid derivatives are the carriers used to deliver other adjuvants which also have weak immunostimulatory properties on their own; squalene is one example.
Adjuvants that are administered separately from the vaccine injection include thymic hormone derivatives, dehydroepiandrosterone (DHEA) and melatonin. The thymic hormone derivatives such as thymosin alpha 1 and thymulin have only mild immunostimulatory effects. They also suffer the disadvantage of being impractical since several injections are required to elicit the desired response.
The use of DHEA and melatonin as adjuvants is new and as yet poorly studied. Both compounds naturally decline with age (as do the thymic hormones), can be administered orally, and appear to be nontoxic. Their effectiveness as adjuvants, however, remains to be determined.
It is clear from this array of information that to date an adjuvant has not been developed that combines the advantages of a high degree of potency, convenience of administration, and the absence of any health risk. Thus, the prior art teaches age-linked diminished immune response and age-linked decrease in oxidative metabolism of fatty acids. The prior art also teaches the use of carnitine to enhance the proliferative response of lymphocytes in vitro. The art does not, however, provide any link between the in vivo administration of L-carnitine to animals (or humans) and enhanced production of antibodies by B-cells in response to concomitant vaccine administration.